1. Field of the Invention
This invention relates generally to methods and compositions useful in inhibiting disorders dependent upon expression of the inducible transcription factor NF-xcexaB.
2. Description of Related Art
The NF-xcexaB transcription activator is a multiprotein complex which can rapidly induce the synthesis of defense and signalling proteins upon exposure of cells to a wide variety of mostly pathogenic agents. Three protein subunits, IxcexaB, p50, and p65, control the biological functions of NF-xcexaB. IxcexaB is a 35-43 kDa subunit which inhibits the DNA-binding of NF-xcexaB and serves to retain NF-xcexaB in an inducible form in the cytoplasm of unstimulated cells. Upon stimulation of cells, IxcexaB dissociates from the inactive complex with p65 and p50. The released p50-p65 complex can then migrate into the nucleus and potently transactivate genes. p50, one of the two DNA-binding subunits of NF-xcexaB, serves to recognize the more highly conserved half site in decameric sequence motifs with the consensus sequence GGGRNNYYCC. p50 has homologies to the rel and dorsal proteins, both of which are also involved in cytoplasmic/nuclear signalling, and is identical to factors known as KBF1, H2TF1 and EBP-1. p50 is synthesized as an inactive precursor of 110 kDA.
The other DNA binding subunit, p65, binds to the less conserved half site of kB motifs and is apparently also homologous to rel/dorsal proteins. The combination of p50 and p65 DNA binding subunits in NF-xcexaB extends the repertoire of binding motifs recognized with high affinity. Only p65 appears to bind IxcexaB. The inducibility of NF-xcexaB is thus equally dependent on the presence of p65 as of IxcexaB. The NF-xcexaB system is so far unique among transcription factors in that the interaction of three distinct subunits control multiple regulatory characteristics of a transcription activator including the subcellular localization and the activity, inducibility, and specificity of DNA binding.
Many different binding sites for NF-xcexaB have been characterized and for most the base requirements for protein binding have been defined by methylation interference analysis or other footprinting techniques (Baeuerle, Biochem Biophys. Act., 1072:63, 1991). Most binding sites are decameric, but some are undeca- or dodecameric. The latter may arise from NF-xcexaB binding in a mutually exclusive manner to two or three decameric motifs within the longer sequences. An alternative explanation is that p50-p65 or the p50 dimer can contact 10, 11 or even 12 base pairs. Support for the latter hypothesis comes from mutational analysis of the enhancer of the MHC class I gene H-2Kb.
Among those disorders which are linked to activation of NF-xcexaB is leukemia caused by the retrovirus HTLV-1. Human T-cell leukemia virus (HTLV-1) is recognized as the etiologic agent of the human malignancy, adult T cell leukemia (B. J. Poiesz, Proc. Natl. Acad. Sci. USA, 77:7415, 1980; D. J. Slamon, et al., Science, 226:61, 1984; W. C. Goh, et al., ibid. 297:1227, 1985).
Circumstantial data has implicated the HTLV-I encoded tax gene in leukemo-genesis. This gene encodes a 40kD protein that causes transcriptional transactivation of viral gene expression and also activates expression of certain cellular genes that are important for growth (A. J. Cann, et al., Nature 318:571, 1985; B. K. Felber, et al., Science, 229:675, 1985; J. Fujisawa, et al., Embo Journal 5:713, 1986). In vitro studies have demonstrated that tax can activate the promoters of the interleukin 2 receptor (IL-2R) a-chain, GM-CSF, fos, PDGF, IL-6, NGF, TGF-xcex2, HIV LTR as well as its own LTR (D. J. Slamon et al., Science, 226:61, 1984; W. C. Goh et al., ibid., 227:1227, 1985; J. Inoue, et al., Embo Journal 5:2883, 1986; S. Miyatake, et al., Mol. Cell. Biol. 8:5581, 1988; K. Nagata, et al., J. Virol. 63:3220, 1989; L. Ratner, Nucleic Acid Research, 17:4101, 1989; J. Sodroski, et al., Science, 228:1430, 1985; J.E. Green, et al., Mol. Cell. Biol. 11:4635, 1991; S. J. Kim, et al., J. Exp. Med., 172:121, 1990; E. Bohnlein, et al., J. Virol., 63, 1578, 1988). In vitro studies of tax effects on gene expression, have demonstrated two independent pathways for its action on transcription (M. R. Smith and W. C. Green, J. Clin. Invest. 87:761, 1991). The first affects the family of nuclear transcription factors related to c-rel which bind to NF-xcexaB sites and are important for the normal activation of lymphocytes. NF-xcexaB response sequences occur in a number of genes including the HIV LTR (G. Nable, et al., Nature London, 326:711, 1987) and the IL-6 promoter (T. A. Libermann, et al., Mol. Cell. Biol., 10:2327, 1990). The heterodimer composed of the p5C and p65 rel related proteins have been shown to affect the transcription of many of these genes (P. A. Baeuerle, Biochem. Biophys. Acta, 1072:63, 1991). The other effect of tax is thought to be NF-xcexaB independent, whereby tax activates its own promoter through three tax responsive elements (TREs). Similar sequence motifs have been identified in fos (M. Fujii et al., Proc. Natl. Acad. Sci., USA, 85:8526, 1988), an immediate early response gene.
Unfortunately, although it is known that tax and other disorders appear linked to activation of NF-xcexaB, no therapeutic modalities exist which can suppress this activation and thereby inhibit progression or establishment of the disorder. The present invention addresses this need and provides composition and means of accomplishing this goal.
The present invention arose from the discovery that an antisense oligonucleotide which hybridizes to nuclear factor-xcexaB (NF-xcexaB) mRNA can be used to suppress processes which depend upon activation of NF-xcexaB.
As a consequence of this discovery, the present invention represents a major improvement over existing techniques for suppressing NF-xcexaB dependent processes which often entail use of chemicals which are highly toxic, especially in terms of their utilization in vivo. The antisense oligonucleotides of the invention display a high degree of biocompatibility with host systems such that the low efficiency and toxic aspects of prior art methodologies and compositions is avoided.